Our overall approach is to kinetically resolve steps in the pathway of viral envelope glycoprotein-mediated membrane fusion and to uncover physical parameters underlying those steps using a variety of biochemical, biophysical, virological, and molecular and cell biological techniques in vitro studies with infectious virus and HIV envelope proteins expressed in cells. We are using peptide-based entry inhibitors linked to specific lipids to probe details of the fusion reaction. We have recently shown that conjugation of sphinganine (a precursor of dehydrospingomyelin) confers a striking specificity to the inhibitory potential of a short HIV-based peptide suggesting that sphingopeptides act as double edged swords with both lipid and peptide playing a role in the inhibition of HIV entry. We have developed novel methodologies to study fusion based on photo-induced chemical reactions in the membrane using hydrophobic probes such as Iodonaphtylazide. We are applying this methodology both in the analytical mode (identification of domains of viral proteins and of receptors involved in fusion) and the functional mode (affecting viral protein-induced fusion). Using photo-reactive hydrophobic probes we have found ways to inactivate viral envelope glycoproteins while leaving their overall structures intact. These studies have important implications for anti-viral therapies and vaccine development.